The primary concentration in this fiscal year has been to investigate the role of the proline-directed kinase, Cdk5, in regulating cell-cell and cell-matrix adhesion. Previous studies of corneal epithelial wound closure indicated that inhibitors of Cdk5 enhance the rate of epithelial cell migration, suggesting that such inhibitors might be therapeutically useful in treating certain corneal epithelial defects. Results of in vivo testing in mice, carried out in collaboration with Dr. Mary Ann Stepp, George Washington University, have shown that dropwise, topical administration of an inhibitor of Cdk5 activity significantly enhances wound closure without increasing inflammation. Moreover, treated corneas showed normal re-epithelialization of the wound 2 and 3 weeks after wounding. Since matrix metalloproteinases (MMPs) play an important role in epithelial cell migration by degrading and remodeling the extracellular matrix, we also examined the effect of the Cdk5 inhibitor on these enzymes. Eighteen hours after debridement wounding, corneas treated with olomoucine showed elevated levels of MMP9 and decreased levels of MMP2, demonstrating that Cdk5 plays a role in regulating expression of these important enzymes. Studies of the role of Cdk5 in regulating E-cadherin junction formation in cornea have shown that Cdk5 activity stabilizes E- cadherin junctions. We have found that suppressing Cdk5 expression in cultured corneal epithelial cells with siRNA reduces the amount of E-cadherin participating in functionsl cell-to-cell junctions. Similarly, expression of a dominant negative mutation of Cdk5 decreases the half-life of E-cadherin and increases the amount of E-cadherin associated with the lysosomal compartment. Finally, pharmacological inhibition of Cdk5 activity reduces the ability of dissociated corneal epithelial cells to attach to a surface coated with E-cadherin, and increases the degradation of surface biotinylated E-cadherin. These results demonstrate that Cdk5 activity is an essential regulator of E-cadherin mediated cell-to-cell junctions in the corneal epithelium, and thus plays an important role in maintaining the integrity of the epithelial cell sheet.[unreadable] Cdk5 is known to regulate cytoskeletal organization in neurons and to interact with a number of cytoskeletal proteins. Work from this laboratory has shown that Cdk5 is also involved in regulating the cytoskeletal architecture of epithelial cells of the lens and cornea. In the past year, studies of the mechanism of this effect have demonstrated that phosphorylation of Cdk5 on Y15 is required for formation of stress fibers in spreading lens epithelial cells. Expression of Cdk5 bearing a mutation at this site blocks stress fiber formation, while expression of wild type Cdk5 or inactive mutations of Cdk5 that are phosphorylated on Y15 do not. In addition, we have demonstrated that suppression of Cdk5 expression with siRNA oligonucleotides leads to clumping of the actin cytoskeleton; in contrast, cells transfected with a scrambled oligonucleotide had normal cytoskeletal architecture. Together these findings demonstrate that Cdk5 is an essential regulator of cytoskeletal organization in lens epithelial cells and suggest that this protein has scaffolding functions that are mediated by phosphorylation of Y15.[unreadable] During the past year we have also initiated pilot studies of two additional signaling pathways implicated in epithelial cell adhesion and migration: selective ubiquitylation by E3 ubiquitin ligases and the Notch pathway. Studies of ubiquitylation have focused on the protein PdLim2, an E3 ubiquitin ligase which is highly expressed in the corneal epithelium. Preliminary results show that PdLim2 associates with the actin cytoskeleton. Studies of Notch signaling seek to determine the role of Notch2 in the lens. Initial results suggest multiple modes of action for this signaling pathway in lens differentiation.